Transplantation of tissue into an animal, such as, for example, stem cells, cells cultured in vitro, or isolated primary cells, typically involves direct introduction of cellular material into the recipient, either into the blood stream or directly into a tissue. However, these procedures are associated with significant complications, such as thrombosis, which reduces cell survival.
Tissue engineering can provide a solution to this problem by providing a three dimensional support that acts as a substrate for cell attachment. It has been previously demonstrated that cells seeded in a properly designed support could recreate the in-vivo microenvironment, thereby facilitating cell-cell interactions and expression of differentiated functions. To construct such complex structures, the efficiency of the cell seeding process can be important to the overall performance of the tissue-engineered construct.
Prior to the present invention, seeding of cells onto supports has involved simple depositing of cells onto the support by relying on passive diffusion of cells into the support. Several other approaches have been developed to enhance the efficiency of cell seeding. For example, spinner flasks have been used in seeding of chondrocytes onto polyglycolic acid supports (Vunjak-Novakovic et al., “Dynamic cell seeding of polymer supports for cartilage tissue engineering,” Biotechnol. Prog. 14(2):193-202, 1998). The procedure involved suspending the supports via needles in a cell suspension and mixing with a magnetic stir bar at 50 rpm. The process required a long time to complete, ranging from several hours to one day.
Another approach for seeding cells is the use of centrifugation, which yields minimum stress to the seeded cells and enhances seeding efficiency. A cell seeding method was developed by Yang et al. (J. Biomed. Mater. Res. 55(3): 379-86, 2001), referred to as Centrifugational Cell Immobilization (CCI). Hepatocytes were seeded onto hydrophilic porous poly (vinyl formal) cubes. Both the cubes and hepatocytes were suspended in media in a centrifugation tube and were exposed to alternating centrifugation and resuspension steps. The procedure yielded 40% seeding efficiency and required a large number of hepatocytes (2-8×107 cells). Dar et al. (Biotechnol. Bioeng. 80(3): 305-12, 2002) utilized a more controlled approach in cell seeding via centrifugation. Cardiomyocytes were seeded onto a hydrophilic alginate support by placing the support into a well of a 96-well plate and pipetting 10 μl of cell suspension onto it. The plate was then placed onto a plate holder-type rotor and centrifuged for 6 minutes at 1000×g, 4° C. A seeding efficiency of 80-90% was reported in an alginate support, which decreased to 60% when higher seeding densities were used per support. The centrifugation methods described above have yielded some success but have limitations. A vital issue in the process is the porosity of the support. The centrifugal force pressures the cell suspension through the support where the cellular material gets entangled within the pores of the support. If the porosity is too large, the cellular material passes all the way through the support to the bottom of the centrifugation chamber leading to a drop in seeding efficiency. On the other hand, lowering the porosity of the support to accommodate this issue may have a negative effect on the survival of the seeded cellular material. A high porosity is essential in allowing diffusion of oxygen and nutrition.
Seeding cells onto a hydrophobic support is usually more complex than onto a hydrophilic one. Cells are usually suspended in a culture media solution with water being the major component. A hydrophobic support repels a cell suspension preventing cells from infiltrating said support. In order to overcome such barrier, a driving force is required. The force regardless of its source exposes the cells to a stress component that is harmful to the cells. Thus, there remains a need to develop a simple and reproducible method to seed cells onto porous supports particularly hydrophobic ones with high seeding efficiency and little or no loss in cell viability.